This application is a 371 of PCT/GB99/00231, filed Jan. 22, 1999, which claims the benefit of United Kingdom 9801398.0, filed Jan. 22, 1998, the disclosures of which are incorporated herein by reference in their entirety.
The present invention relates to compounds capable of acting both as sources of nitric oxide (NO-donors) and as scavengers of superoxide ion (O2xe2x88x92) (superoxide dismutase (SOD)-mimetic).
Many disease states, including diabetes mellitus and various cardiovascular diseases, are associated with oxidative stress and endothelial dysfunction. In particular, endothelial dysfunction is the hallmark of the pathophysiological process (atherosclerosis) leading to a spectrum of clinically related diseases of the cardiovascular system (e.g. from stable angina pectoris to myocardial infarction (MI) and congestive heart failure), as well as being the primary event in pathologies relating to other body systems involving superoxide and/or other reactive oxygen species (ROS).
For over a century, nitroglycerin (GTN) has been the drug of choice for the treatment of various types of myocardial ischemia (angina), including myocardial infarction, and a mainstay in the treatment of other heart diseases with and without ischemic etiology (congestive heart failure, isolated systolic and resistant hypertension). Because of its pathogenic nature (chronicity with acute exacerbation), prophylactic and acute treatments are necessary to prevent complications with potentially fatal outcomes ( greater than 25% death for acute MI).
However, the phenomenon of tolerance to the anti-anginal effects of GTN and to all other existing organic nitrates is of a special clinical significance. In particular, early development of tolerance to the drug is by far the most serious drawback of nitrate therapy, especially during acute myocardial infarction.
Evidence has been provided to support an involvement of the superoxide anion in the mechanism/s underlying nitrate tolerance and cross-tolerance (Munzel et al., J. Clin. Invest. 95, 187-194 (1995)). According to this report, increased levels of superoxide anion were found to accompany tolerance development to GTN in vascular tissue after in vivo administration of the drug. Treatment with superoxide dismutase (SOD) significantly enhanced maximal relaxation of control and tolerant vascular tissue to GTN and other exogenous and endogenous vasodilators. This can be explained on the basis of an enhancing effect of superoxide on the induction of tolerance (pseudo-tolerance). It is believed that if the normally tightly controlled balance between nitric oxide (NO) and superoxide (O2xe2x88x92) in the vascular wall is disturbed, elevated levels of superoxide anion prevail, inactivating NO and furthermore generating toxic peroxynitrite. The protective effect of superoxide dismutase against nitrate tolerance has been demonstrated in vitro (Jia et al., J. Pharmacol. Exp. Ther., 267,371-378 (1993) and Kowaluk et al., J. Pharmacol. Exp. Ther., 255, 109-144 (1990)). However, being an enzyme, SOD is practically cell impermeable and cannot therefore be used therapeutically. There remains therefore a need for a nitrate-based therapy which does not suffer from the problem of nitrate tolerance.
According to the present invention there is provided a chemical compound comprising a nitric oxide (NO) donor and a superoxide (O2xe2x88x92) scavenger.
The nitric oxide donor may comprise any group capable of acting as a source of nitric oxide (NO). Preferably, the nitric oxide donor is an xe2x80x94ONO2 group. The superoxide scavenger may comprise any group capable of acting as a scavenger of superoxide (O2xe2x88x92). Preferably, the superoxide scavenger is a nitroxide free radical (Nxe2x86x92O.) group.
The compounds of the present invention may comprise one or more NO donors and one or more superoxide scavengers.
The compounds of the present invention not only provide a source of nitric oxide but in acting as an antioxidant scavenger of superoxide anion give rise to both a direct benefit derived from removal of injurious superoxide anion and a benefit in protecting both ambient and endogenous and liberated exogenous NO from inactivation by superoxide anion.
Without prejudice to the scope of the present invention, it is believed that oxidative damage is mediated by intracellular redox-active metal reactions catalyzed by highly reactive oxygen species (i.e. hydroxyl radicals). The generation of such reactive oxygen products depends on the availability of their common precursor, the superoxide anion. Mitochondria, microsomes and other various enzyme systems are known to produce superoxide anion that reacts with nitric oxide at or near diffusion controlled rates to form the powerful oxidant peroxynitrite. At pH 7.4, peroxynitrite protonates to form peroxynitrous acid (pKa 6.6) which decays homolytically to form hydroxyl and nitrogen dioxide radicals in addition to a host of other ions. The extent to which these later reactive ions and radicals can cause cellular damage and death depends on the rate of formation of their peroxynitrite precursor. Under control (non-diseased) conditions, where the levels of NO are found in equilibrium between rates of NO-synthesis and degradation, the rate of formation of this peroxynitrite precursor is thought to depend solely on the levels of the superoxide anion. This is particularly important in cases involving xe2x80x9cbelow-normalxe2x80x9d levels of xe2x80x9cbiologically activexe2x80x9d NO, such as in diseases for which therapeutic intervention with exogenous NO-donors is clinically indicated. Consequently, the extra- and intra-cellular activity of the enzyme SOD must have a cardinal role in maintaining cellular survival, tissue integrity and adequately balanced physiological function.
Since superoxide anion is an available and continuously-formed by-product generated through normal metabolic processes, and since its elimination is mediated either by dismutation by the enzyme SOD or via its reaction with NO to form the potentially hazardous peroxynitrite, it is now believed that the ultimate means by which a modification and/or treatment of xe2x80x98pathological processesxe2x80x99 involving imbalanced ratio of NO to superoxide is by an intervention with therapeutic agents capable of simultaneously and favourably affecting both components; the NO and O2xe2x88x92.
By virtue of the NO donor and superoxide scavenging activities being physically linked in the same molecule, the compounds of the present invention ensure that an increase in the level of NO is accompanied by reduced levels of superoxide thereby avoiding high levels of peroxynitrite and oxidant metabolites thereof. Preferred compounds according to the present invention are of the formula:
Preferred compounds according to the present invention are of the formula: 
wherein
R1 may be the same or different and are independently selected from hydrogen, alkoyl, alkoxy, carboxy, hydroxy, amino, amido, cyano, nitro, thio, sulphonyl, sulphoxide, alkyl groups and groups comprising an NO-donor, provided that at least one R1 is a group comprising an NO-donor group;
R2 may the same or different and are independently selected from alkyl groups;
n is an integer 1, 2 or 3;
or a salt thereof
The groups R1 may be the same or different and are independently selected from hydrogen, alkoyl, alkoxy, carboxy, hydroxy, amino, amido, cyano, nitro, thio, sulphonyl, sulphoxide, alkyl groups and groups comprising an NO-donor group, provided that at least one R1 is a group comprising an NO-donor. R1 groups which do not comprise an NO-donor group are preferably hydrogen or alkyl groups. The or each R1 group comprising an NO-donor group may comprise one or more NO-donor groups. Preferably, the or each NO-donor group is an xe2x80x94ONO2 group.
The or each R1 group comprising an NO-donor group may comprise an NO-donor group alone or may comprise an NO-donor group linked via a C1 to C20 alkylene chain optionally comprising one or more heteroatoms. The alkylene chain may be branched or unbranched, cyclic or acyclic, saturated or unsaturated, where cyclic the alkylene chain is preferably C3 to C12, more preferably C5 to C10, more preferably C5 to C7. Where acyclic, the alkylene chain is preferably C1 to C16, more preferably C1 to C6. The alkylene chain may be unsubstituted or substituted as defined hereinbelow in respect of alkyl groups. The alkylene chain may comprise one or more heteroatoms, for example nitrogen, oxygen or sulphur atoms which may interupt the alkylene chain or may link the alkylene chain to the NO-donor group or to the rest of the molecule (such that the R1 group comprising the NO-donor group is linked to the rest of the molecule via a heteroatom, such as an oxygen (e.g. ether linkage), nitrogen (e.g. amino or amido linkage) or sulphur (e.g. thioether linkage)).
Preferred R1 groups comprising NO-donors comprise groups of the formula:
xe2x80x94Xxe2x80x94Yxe2x80x94ONO2
where X is absent or is O, NH or S; and
Y is a C1-20 alkylene chain
Preferably, X is O, NH or S.
In a preferred embodiment, at least one R1 is a polar or ionisable group such as carboxyl or amino, which improves the solubility of the compound.
The groups R2 may be the same or different and are selected from alkyl groups. Preferably, the groups R2 are each methyl groups. In an alternative preferred embodiment, one or more of the groups R2 is a lipophilic group, such as a substituted or unsubstituted C1 to C8 alkyl group, capable of assisting in transport of the compound across the blood-brain membrane.
n may be 1, 2 or 3. Preferably n is 1 or 2, More preferably n is 2.
As used here reference to alkyl groups means a branched or unbranched, cyclic or acyclic, saturated or unsaturated (e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, the alkyl group is preferably C3 to C12, more preferably C5 to C10, more preferably C5 to C7. Where acyclic, the alkyl group is preferably C1 to C16, more preferably C1 to C6, more preferably methyl. Reference in the present specification to an alkoxy group means an alkyl-O-group. Reference to alkoyl group means an alkyl-CO-group.
The alkyl groups may be substituted or unsubstituted, preferably unsubstituted. Where substituted, there will generally be 1 to 3 substituents present, preferably 1 substituent Substituents may include halogen atoms and halomethyl groups such as CF3 and CCl3; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl and aryloyloxy; nitrogen containing groups such as amino, amido, alkylamino, dialkylamino, cyano, azide, nitrato and nitro; sulphur containing groups such as thiol, alkylthiol, sulphonyl and sulphoxide; heterocyclic groups containing one or more, preferably one, heteroatom, such as thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, benzofuranyl, isobenzofuryl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl, isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl and carbolinyl; alkyl groups, which may themselves be substituted; and aryl groups, which may themselves be substituted, such as phenyl and substituted phenyl. Alkyl includes substituted and unsubstituted benzyl.
Reference to amino includes substituted or unsubstituted amino.
Reference in the present specification to halogen means a fluorine, chlorine, bromine or iodine radical, preferably fluorine or chlorine radical.
The present invention extends to dimers and higher multimers of the compounds of the present invention; for example dimers of compounds of the present invention linked via R1 groups (e.g. via an alkylene chain therein).
Particularly preferred compounds according to the present invention comprise:
4-Nitrato-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO-4-mononitrate)
3,4-Dinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical (TEMPO-3,4-dimitrate)
3,4,5-Trinitrato-2,2,6,6-Tetramethylpiperidinyloxy, free radical (TEMPO-3,4,5-trinitrate)
4-(2,3-Dinitrato-Prop-1-oxy)-2,2,6,6-Tetramethyl-piperidinyloxy, free radical (4-(2,3-dinitrato-prop-1-oxy) TEMPO)
1,3-di (4-Oxy-TEMPO)2-Nitrato-Propane
1,4-di (4-Oxy-TEMPO)-2,3-Dinitrato-Butane
3-Nitrato-4-carboxy-2,2,6,6-tetramethylpiperidinyloxy, free radical (TEMPO-4-carboxyl-3-nitrate)
4-Nitrato-3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolidin-1-yloxy, free radical (PROXYL-3-carbamoyl-4-nitrate)
The present invention further extends to methods of synthesising the compounds of the present invention. The compounds of the present invention may be synthesised according to the following generalised reaction schemes. It will be appreciated that the reaction schemes are merely illustrative of generally applicable procedures which may be modified as appropriate to produce the compounds of the present invention. In the following reaction schemes the 2,2,6,6-tetra substitution present in, for example TEMPO compounds is, for simplicity, not shown. 
According to a further aspect of the present invention there is provided a compound according to the present invention for use in a method of treatment.
The compounds of the present invention may be employed in the treatment of any condition associated with endothelial dysfunction or oxidative stress including diabetes mellitus, cardiovascular diseases (such as ischaemic heart disease, angina pectoris, myocardial infarction, congestive heart failure, atherosclerosis, hypertension and arrhythmia), asthma, trauma, shock (hypovolumic, neurogenic or septic), neurotoxicity, neurodegenerative and neurological disorders (including Alzheimer and Parkinson""s diseases, amyotrophic lateral sclerosis, multiple sclerosis, convulsive (seizure) disorders, AIDS-dementia and disorders which involve processes of learning and memory), disorders of gastric secretions, relaxation and peristalsis of the intestinal tract (including sphincters), drug and disease-induced nephropathies, pathological (premature) and physiological uterine contractions, cellular defense impairment, endothelial dysfunction-induced diseases and insulin-resistance in diabetes, pregnancy-induced hypertension, chemotaxis and phagocytic impairment in immunological disorders, cerebrovascular diseases, aggregation disorders, fertility and reproductive disorders (e.g. penile erection and treatment of male impotence).
According to a further aspect of the present invention there is provided use of a compound according to the present invention for use in the manufacture of a medicament for treating a condition associated with oxidative stress or endothelial dysfunction, preferably diabetes mellitus or cardiovascular disease.
According to a further aspect of the present invention there is provided a method of treating a disease associated with oxidative stress or endothelial dysfunction (such as diabetes mellitus or cardiovascular disease) comprising administering to a patient in need of such treatment an effective dose of a compound of the present invention
According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a compound of the present invention in combination with a pharmaceutically acceptable excipient.
Compounds of the present invention may be administered in a form suitable for oral use, for example a tablet, capsule, aqueous or oily solution, suspension or emulsion; for topical use including transmucosal and transdermal use, for example a cream, ointment, gel, aqueous or oil solution or suspension, salve, patch, plaster or as a component of a lubricant for a condom; for nasal use, for an example a snuff, nasal spray or nasal drops; for vaginal or rectal use, for example a suppository; for administration by inhalation, for example a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, for example a tablet or capsule; or for parenteral use (including intravenous, subcutaneous, intramuscular, intravascular or infusion), for example a sterile aqueous or oil solution or suspension, or incorporated in a biodegradable polymer. In general the above compositions may be prepared in a conventional manner using convention excipients, using standard techniques well known to those skilled in the art of pharmacy. Preferably, the compound is administered orally or topically.
For oral administration, the compounds of the invention will generally be provided in the form of tablets or capsules or as an aqueous solution or suspension.
Tablets for oral use may include the active ingredient mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, the compounds of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer""s solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate. The compounds of the invention may also be provided in a biodegradable polymer, for example for use in conjunction with stents in angioplasty (e.g. adsorbed on a stent or applied directly to the site of the procedure for slow release of the active agent).
It will be appreciated that the dosage levels used may vary over quite a wide range depending upon the compound used, the severity of the symptoms exhibited by the patient and the patient""s body weight. Without limitation to the present invention, typical dosages for example for the treatment of angina may be of the order of 1 to 100 mg, preferably 5 to 40 mg, given two or three times daily or 1 to 200 mg, preferably 20 to 50 mg, in sustained release formulations given once or twice daily. Typical dosages for example for acute myocardial infarction may be of the order of 0.1 to 10 mg, preferably 1 to 2 mg, sublingually; 0.5 to 50 mg, preferably 5 to 10 mg, orally; or 1 to 100 micrograms, preferably 10 to 20 micrograms, per minute intravenously.